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The Clp protease system is a promising, noncanonical drug target against Mycobacterium tuberculosis (Mtb). Unlike in Escherichia coli , the Mtb Clp protease consists of two distinct proteolytic subunits, ClpP1 and ClpP2, which hydrolyze substrates delivered by the chaperones ClpX and ClpC1. While biochemical approaches uncovered unique aspects of Mtb Clp enzymology, its essentiality complicates in vivo studies. To address this gap, we leveraged new genetic tools to mechanistically interrogate the in vivo essentiality of the Mtb Clp protease. While validating some aspects of the biochemical model, we unexpectedly found that only the proteolytic activity of ClpP1, but not of ClpP2, is essential for substrate degradation and Mtb growth and infection. Our observations not only support a revised model of Mtb Clp biology, where ClpP2 scaffolds chaperone binding while ClpP1 provides the essential proteolytic activity of the complex; they also have important implications for the ongoing development of inhibitors toward this emerging therapeutic target.

The Clp protease system is a promising, noncanonical drug target against

Phosphopantetheinyl hydrolase, PptH (Rv2795c), is a recently discovered enzyme from Mycobacterium tuberculosis that removes 4′-phosphopantetheine (Ppt) from holo-carrier proteins (CPs) and thereby opposes the action of phosphopantetheinyl transferases (PPTases). PptH is the first structurally characterized enzyme of the phosphopantetheinyl hydrolase family. However, conditions for optimal activity of PptH have not been defined, and only one substrate has been identified. Here, we provide biochemical characterization of PptH and demonstrate that the enzyme hydrolyzes Ppt in vitro from more than one M. tuberculosis holo-CP as well as holo-CPs from other organisms. PptH provided the only detectable activity in mycobacterial lysates that dephosphopantetheinylated acyl carrier protein M (AcpM), suggesting that PptH is the main Ppt hydrolase in M. tuberculosis. We could not detect a role for PptH in coenzyme A (CoA) salvage, and PptH was not required for virulence of M. tuberculosis during infection of mice. It remains to be determined why mycobacteria conserve a broadly acting phosphohydrolase that removes the Ppt prosthetic group from essential CPs. We speculate that the enzyme is critical for aspects of the life cycle of M. tuberculosis that are not routinely modeled. IMPORTANCE Tuberculosis (TB), caused by Mycobacterium tuberculosis, was the leading cause of death from an infectious disease before COVID, yet the in vivo essentiality and function of many of the protein-encoding genes expressed by M. tuberculosis are not known. We biochemically characterize M. tuberculosis’s phosphopantetheinyl hydrolase, PptH, a protein unique to mycobacteria that removes an essential posttranslational modification on proteins involved in synthesis of lipids important for the bacterium’s cell wall and virulence. We demonstrate that the enzyme has broad substrate specificity, but it does not appear to have a role in coenzyme A (CoA) salvage or virulence in a mouse model of TB.

Nonribosomal peptide synthetases (NRPSs) underlie the biosynthesis of many natural products that have important medicinal utility. Protection of the NRPS peptide products from proteolysis is critical to these pathways and is often achieved by structural modification, principally the introduction of d-amino acid residues into the elongating peptide. These amino acids are generally formed in situ from their l-stereoisomers by epimerization domains or dual-function condensation/epimerization domains. In singular contrast, the thioesterase domain of nocardicin biosynthesis mediates both the effectively complete l- to d-epimerization of its C-terminal amino acid residue (≥100:1) and hydrolytic product release. We report herein high-resolution crystal structures of the nocardicin thioesterase domain in ligand-free form and reacted with a structurally precise fluorophosphonate substrate mimic that identify the complete peptide binding pocket to accommodate both stereoisomers. These structures combined with additional functional studies provide detailed mechanistic insight into this unique dual-function NRPS domain., NocTE is a nonribosomal peptide synthetase thioesterase that completes the biosynthesis of pro-nocardicin G, the precursor for nocardicin β-lactam antibiotics. Here the authors provide mechanistic insights into NocTE by determining its crystal structures in the ligand-free form and covalently linked to a fluorophosphonate substrate mimic.

An efficient route to substituted N-fused aromatic heterocycles, including indolizines, imidazo[1,2-a]pyridines, and imidazo[1,5-a]pyridines from azole aldehydes is reported. A three-component Wittig olefination of the aldehydes with fumaronitrile and triethylphosphine affords predominantly E-alkenes that undergo rapid cyclization upon in situ treatment with catalytic amounts of mild base. Substituent control of the 1-, 2-, and 3-positions of the resulting heteroaromatic bicycles is shown. Alternatively, the isolable E-alkene product from pyrrole-2-carboxaldehyde, under dianionic conditions, undergoes selective alkylation with various electrophiles, followed by in situ annulation to indolizines additionally substituted at the 6-position.

As a growing number of clinical isolates of Mycobacterium abscessus are resistant to most antibiotics, new treatment options that are effective against these drug-resistant strains are desperately needed. The majority of the linkages in the cell wall peptidoglycan of M. abscessus are synthesized by nonclassical transpeptidases, namely, the l , d -transpeptidases. Emerging evidence suggests that these enzymes represent a new molecular vulnerability in this pathogen. Recent studies have demonstrated that inhibition of these enzymes by the carbapenem class of β-lactams determines their activity against Mycobacterium tuberculosis . Here, we studied the interactions of β-lactams with two l , d -transpeptidases in M. abscessus , namely, Ldt Mab1 and Ldt Mab2 , and found that both the carbapenem and cephalosporin, but not penicillin, subclasses of β-lactams inhibit these enzymes. Contrary to the commonly held belief that combination therapy with β-lactams is redundant, doripenem and cefdinir exhibit synergy against both pansusceptible M. abscessus and clinical isolates that are resistant to most antibiotics, which suggests that dual-β-lactam therapy has potential for the treatment of M. abscessus . Finally, we solved the first crystal structure of an M. abscessus l , d -transpeptidase, Ldt Mab2 , and using substitutions of critical amino acids in the catalytic site and computational simulations, we describe the key molecular interactions between this enzyme and β-lactams, which provide an insight into the molecular basis for the relative efficacy of different β-lactams against M. abscessus .

Summary Diphenylphosphonates (DPPs) have been used for 50 years to inactivate serine hydrolases, creating adducts representative of tetrahedral intermediates of this important class of enzymes. Failure to react at active site serine residues, however, can thwart their usefulness. Here, we describe a facile route and allied mechanistic studies to highly reactive, structurally complex organofluorophosphonates (FPs) by direct fluorinative hydrolysis of DPPs. Advantages over current preparations of FPs are exemplified by the synthesis of a β-lactam-containing peptide substrate analog capable of modifying the C-terminal, dual-function thioesterase involved in nocardicin A biosynthesis. Although this serine hydrolase was found to resist modification by classic DPP inhibitors, active site selective phosphonylation by the corresponding FP occurs rapidly to form a stable adduct. This simple, late-stage method enables the ready preparation of FP probes that retain important structural motifs of native substrates, thus promoting efforts in mechanistic enzymology by accessing biologically relevant enzyme-inhibitor co-structures.

Starting from pyrrole- and imidazolecarboxaldehydes, Wittig olefination or alkylation with fumaronitrile and sequential cycloaromatization leads to indolizines and imidazopyridines, respectively.